Nitroxyl progenitors in the treatment of heart failure

ABSTRACT

Administration of an HNO/NO −  donating compound, such as Angeli&#39;s salt, increases myocardial contractility while concomitantly lowering left ventricular preload in subjects experiencing heart failure. Moreover, administration of the HNO/NO −  donating compound isopropylamine (IPA)/NO (Na(CH 3 ) 2 CHNHN(O)NO) surprisingly exhibited positive inotropic effects in subjects experiencing heart failure that were superior to those caused by the HNO/NO −  donating compound Angeli&#39;s salt. Additionally, in contrast to the effects observed with NO· donors, administration of an HNO/NO −  donor in combination with a positive inotropic agent did not impair the positive inotropic effect of the positive inotropic agent. Further, HNO/NO −  exerts its positive inotropic effect independent of the adrenergic system, increasing contractility even in subjects receiving beta-antagonist therapy.

FIELD

Pharmaceutical compounds and compositions are disclosed that are usefulto treat heart failure.

BACKGROUND

Congestive heart failure (CHF) is a generally progressive, lifethreatening condition in which myocardial contractility is depressedsuch that the heart is unable to adequately pump the blood returning toit, also referred to as decompensation. Symptoms include breathlessness,fatigue, weakness, leg swelling, and exercise intolerance. On physicalexamination, patients with heart failure often have elevated heart andrespiratory rates (an indication of fluid in the lungs), edema, jugularvenous distension, and enlarged hearts. The most common cause of CHF isatherosclerosis, which causes blockages in the coronary arteries thatprovide blood flow to the heart muscle. Ultimately, such blockages maycause myocardial infarction with subsequent decline in heart functionand resultant heart failure. Other causes of CHF include valvular heartdisease, hypertension, viral infections of the heart, alcoholconsumption, and diabetes. Some cases of CHF occur without clearetiology and are called idiopathic. The effects of CHF on a subjectexperiencing the condition can be fatal.

There are several types of CHF. Two types of CHF are identifiedaccording to which phase of the cardiac pumping cycle is more affected.Systolic heart failure occurs when the heart's ability to contractdecreases. The heart cannot pump with enough force to push a sufficientamount of blood into the circulation leading to a reduced leftventricular ejection fraction. Lung congestion is a typical symptom ofsystolic heart failure. Diastolic heart failure refers to the heart'sinability to relax between contractions and allow enough blood to enterthe ventricles. Higher filling pressures are required to maintaincardiac output, but contractility as measured by left ventricularejection fraction is typically normal. Swelling (edema) in the abdomenand legs is a typical symptom of diastolic heart failure.

CHF is also classified according to its severity. The New York HeartAssociation classification classifies CHF into four classes:

Class I—no obvious symptoms, with no limitations on physical activity;

Class II—some symptoms during or after normal activity, with mildphysical activity limitations;

Class III—symptoms with less than ordinary activity, with moderate tosignificant physical activity limitations;

Class IV—significant symptoms at rest, with severe to total physicalactivity limitations.

Typically, a subject progresses through the classes as the subject liveswith the condition.

Although CHF is generally thought of as a chronic, progressivecondition, it can also develop suddenly. This type of CHF is calledacute CHF, and it is a medical emergency. Acute CHF can be caused byacute myocardial injury that affects either myocardial performance, suchas myocardial infarction, or valvular/chamber integrity, such as mitralregurgitation or ventricular septal rupture, which leads to an acuterise in left ventricular and diastolic pressure resulting in pulmonaryedema, and dyspnea.

Common treatment agents for CHF include, vasodilators (drugs that dilateblood vessels), positive inotropes (drugs that increase the heart'sability to contract), and diuretics (drugs to reduce fluid).Additionally, beta-antagonists (drugs that antagonize beta-adrenergicreceptors) have recently become standard agents for treating mild tomoderate heart failure. Lowes et al., Clin. Cardiol., 23:III11-6 (2000).

Positive inotropic agents include beta-adrenergic agonists, such asdopamine, dobutamine, dopexamine, and isoproterenol. Dobutamine iscommonly given to subjects experiencing late-stage heart failurecharacterized by severely reduced ventricular ejection fraction or theinability of the subject to undertake physical activity withoutdiscomfort. Dobutamine is particularly effective for treating this typeof heart failure because of its cardio-selectivity. U.S. Pat. No.4,562,206 describes dobutamine's cardio-selectivity for the beta-1adrenergic receptor relative to its activity at the vascular alpha andbeta-2 adrenergic receptors. This cardio-selectivity results in adesired positive inotropic effect without a substantial, concomitantincrease or decrease in blood pressure. Such blood pressure changes insubjects experiencing heart failure could cause further deterioration inheart function.

However, the use of beta-agonists has potential complications, such asarrhythmogenesis and increased oxygen demand by the heart. Additionally,the initial short-lived improvement of myocardial contractility affordedby these drugs is followed by an accelerated mortality rate resultinglargely from a greater frequency of sudden death. Katz, HEART FAILURE:PATHOPHYSIOLOGY, MOLECULAR BIOLOGY AND CLINICAL MANAGEMENT, Lippincott,Williams & Wilkins (1999).

Beta-antagonists antagonize beta-adrenergic receptor function. Whileinitially contra-indicated in heart failure, they have been found toprovide a marked reduction in mortality and morbidity in clinicaltrials. Bouzamondo et al., Fundam. Clin. Pharmacol., 15:95-109 (2001).Accordingly, they have become an established therapy for heart failure.Bouzamondo, supra. However, even subjects that improve underbeta-antagonist therapy may subsequently decompensate and require acutetreatment with a positive inotropic agent. Unfortunately, as their namesuggests, beta-antagonists block the mechanism of action of the positiveinotropic beta-agonists that are used in emergency care centers. Bristowet al., J. Card. Fail., 7:8-12 (2001).

Additionally, vasodilating agents are also used to treat heart failure.Vasodilators, such as nitroglycerin, have been used for a long period oftime to treat heart failure. However, the cause of nitroglycerin'stherapeutic effect was not known until late in the last century when itwas discovered that the nitric oxide molecule (NG) was responsible fornitroglycerin's beneficial effects. In fact, the Nobel Prize was awardedin 1998 to three researchers who discovered NO·'s beneficial effects.Opie & White in NITRATES IN DRUGS FOR THE HEART, W. B. Saunder,Philadelphia, 33-53 (2001), explain that such compounds are useful fortreating heart failure due to their balanced venous and arterialvasorelaxant effects. U.S. Pat. No. 5,212,204 describes a group of NO·donating compounds containing the NONO group. The patent discloses thatNO· donated from such compounds has vasodilative properties and can beuseful to treat cardiac diseases that would respond favorably to adecrease in blood pressure, including acute congestive heart failure.The patent identifies Angeli's salt (sodium trioxodinitrate or Na₂N₂O₃)as such a compound. Angeli's salt is a compound that can decompose todonate either NO⁻ or NO· depending on the oxidation state of theenvironment. Fitzhugh & Keefer, Free Radical Biology & Medicine,28(10):1463-1469 (2000). For example, in the presence of oxidants suchas ferricyanide, Angeli's salt decomposes to donate NO·.

Fitzhugh & Keefer, supra.

In some subjects experiencing heart failure, a nitric oxide donor isadministered in combination with a positive inotropic agent to bothcause vasodilation and to increase myocardial contractility. However,this combined administration can impair the effectiveness of positiveinotropic treatment agents. For example, Hart et al., Am. J. Physiol.Heart Circ. Physiol., 281:146-54 (2001) reported that administration ofthe nitric oxide donor sodium nitroprusside, in combination with thepositive inotropic, beta-adrenergic agonist dobutamine, impaired thepositive intotropic effect of dobutamine. Hare et al., Circulation,92:2198-203 (1995) also disclosed the inhibitory effect of NO· on theeffectiveness of dobutamine.

Researchers have also investigated other forms of nitric oxide todetermine their effects on the heart. The nitroxyl species includes thenitroxyl anion (NO⁻), which is the one-electron reduction product ofNO·. Depending on the pH of the environment, the nitroxyl anion may beprotenated to HNO. Experiments testing the effects of NO⁻ donors incardiac diseases have demonstrated that NO⁻ can have a deleteriouseffect on the myocardium when given to reperfused myocardium. In fact,Ma et al., Proc. Nat'l Acad. Sci., 96(25):14617-14622 (1999) reportedthat administration of Angeli's salt as an NO⁻ donor to anesthetizedrabbits 5 minutes prior to reperfusion (after ischemia) increasedmyocardial ischemia/reperfusion injury. Also, Takahira et al., FreeRadical Biology & Medicine, 31(6):809-815 (2001) reported thatadministration of Angeli's salt as an NO⁻ donor during ischemia and 5minutes before reperfusion of rat renal tissue contributed to neutrophilinfiltration into the tissue, which is believed to causeischemia/reperfusion injury.

Patent Cooperation Treaty (PCT) international application PCT/US00/12957discloses administering a charged nitric oxide species to offset theadverse effects of a potassium channel activator in a method ofadministering a potassium channel activator to prevent or treatcardiovascular disorders including, among others, congestive heartfailure. The only NO⁻ donors described in the application arethionitrates that form disulfide species.

SUMMARY

The inventors discovered that administration of a nitroxyl (HNO/NO⁻)donating compound, such as Angeli's salt, increased myocardialcontractility while it concomitantly lowered left ventricular preload insubjects experiencing heart failure. Moreover, administration of theHNO/NO⁻ donating compound isopropylamine (IPA)/NO (Na(CH₃)₂CHNHN(O)NO)surprisingly exhibited positive inotropic effects in subjectsexperiencing heart failure that were superior to those caused by theHNO/NO⁻ donating compound Angeli's salt. Additionally, in contrast tothe effects observed with NO· donors, administration of an HNO/NO⁻ donorin combination with a positive inotropic agent did not impair thepositive inotropic effect of the positive inotropic agent. Further, theinventors discovered that HNO/NO⁻ exerts its positive inotropic effectindependent of the adrenergic system, increasing contractility even insubjects receiving beta-antagonist therapy.

Accordingly, due to their concomitant positive inotropic/lusotropicaction and unloading effects, HNO/NO⁻ donors are helpful in treatingcardiovascular diseases characterized by high resistive load and poorcontractile performance. In particular, HNO/NO⁻ donating compounds suchas IPA/NO are useful treatment agents for heart failure. Moreover, theseagents are useful when used in combination with other positive inotropicagents, such as beta-adrenergic agonists for example, dobutamine.Additionally, HNO/NO⁻ donors are useful for treating heart failure insubjects receiving beta-antagonist therapy.

Provided herein are methods of treating heart failure by administering atherapeutically effective dose at least one HNO/NO⁻ donating compound toa subject experiencing heart failure. Also provided are methods ofadministering a therapeutically effective dose of at least one HNO/NO⁻donating compound in combination with at least one other positiveinotropic agent to a subject experiencing heart failure. Furtherprovided are methods of administering a therapeutically effective doseof at least one HNO/NO⁻ donating compound to a subject who is receivingbeta-antagonist therapy and who is experiencing heart failure.

More particularly, methods are provided herein for administeringcompounds containing the N-oxy-N-nitroso group (diazeniumdolates), whichdonate HNO/NO⁻, to treat heart failure. Such compounds include Angeli'ssalt, IPA/NO, and analogs and derivatives of such compounds.Additionally, methods are provided herein for administering suchcompounds in combination with beta-adrenergic agonists to treat heartfailure. Such agonists include dopamine, dobutamine, and isoproterenol,and analogs and derivatives of such compounds. Also provided are methodsof administering HNO/NO⁻ donors to subjects receiving treatment withbeta-antagonizing agents such as propranolol, metoprolol, bisoprolol,bucindolol, and carvedilol. Further, methods are provided herein fortreating specific classifications of heart failure, such as Class IIIheart failure and acute heart failure.

These and other features and aspects of the disclosed methods willbecome more apparent and better understood with regard to the followingfigures and description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the percentage change from a baseline for severaldiagnostic tests of hearts of conscious dogs experiencing congestiveheart failure (CHF) resulting from administration of the HNO/NO⁻donating compounds Angeli's salt (AS) and isoproylamine/NO (IPA/NO), aswell as the NO· donors diethylamine/NO (DEA/NO) and nitroglycerin (NTG).The diagnostic tests included end-systolic elastance (Ees),preload-normalized maximal change in pressure over change in time dP/dt(D_(EDV)), pre-load recruitable stroke work (PRSW), the time constant ofventricular relaxation (tau), end systolic pressure (Pes), end diastolicvolume (Edv), arterial resistance (Ea), and end diastolic pressure(Edp).

FIG. 2 shows the percentage change from a baseline for severaldiagnostic tests of hearts of conscious dogs experiencing CHF resultingfrom the administration of the HNO/NO⁻ donating compound Angeli's salt(AS), and the administration of AS when the dog hearts were underloading conditions (AS+volume). The diagnostic tests included Ees,preload-normalized maximal change in pressure over change in time dP/dt(dPdt-EDV), PRSW, tau, end systolic pressure (ESP), end systolic volume(ESV), end diastolic volume (EDV), Ea, and end diastolic pressure (EDP).

FIG. 3 shows the percentage change from a baseline for severaldiagnostic tests of hearts of conscious dogs experiencing CHF resultingfrom the administration of the NO· donating compound DEA/NO and theadministration of DEA/NO when the dog hearts were under a load (DEA/NOwith volume). The diagnostic tests included Ees, D_(EDV), PRSW, tau,ESP, ESV, EDV, Ea, and EDP.

FIG. 4 shows the percentage change from a baseline for severaldiagnostic tests of hearts of conscious dogs experiencing CHF resultingfrom the administration of the NO· donating compound nitroglycerin (NTG)and the administration of NTG when the dog hearts were under a load(volume loading). The diagnostic tests included Ees, D_(EDV), PRSW, tau,ESP, ESV, EDV, Ea, and EDP.

FIG. 5 shows the percentage change from a baseline for severaldiagnostic tests of hearts of conscious dogs experiencing CHF resultingfrom the administration of the positive inotropic agent dobutamine (DOB)in combination with the HNO/NO⁻ donating compounds AS and IPA/NO and theNO· donating compounds DEA/NO and NTG. The diagnostic tests includedEes, D_(EDV), PRSW, tau, Pes, ESV, EDV, arterial resistance (EA), andEDP.

FIG. 6 shows the percentage change from a baseline for severaldiagnostic tests of hearts of conscious dogs experiencing heart failure(HF) resulting from the administration of calcitonin gene-relatedpeptide₈₋₃₇ (CGRP₈₋₃₇), administration of CGRP₈₋₃₇ in combination withAS, and administration of AS alone. The diagnostic tests included Ees,D_(EDV), PRSW, tau, Pes, end diastolic pressure (Ped), end diastolicvolume (Ved), and Ea.

FIG. 7 shows the blood plasma CGRP levels in picomoles (pmol) permilliliter (ml) in the artery, vein, and coronary sinus of normalconscious dogs (controls) and conscious dogs experiencing heart failure.

FIG. 8 shows the percentage change from a baseline for severaldiagnostic tests of hearts of normal conscious dogs, which dogs wereunder beta-antagonist therapy with propranolol, resulting fromadministration of the HNO/NO⁻ donating compound AS. The tests includedEes, D_(EDV), and PRSW.

DETAILED DESCRIPTION

Disclosed herein is a method of treating CHF by administering atherapeutically effective dose of at least one nitroxyl (HNO/NO⁻)donating compound to a subject experiencing heart failure. In particularembodiments the HNO/NO⁻ donating compound is IPA/NO. In other particularembodiments the HNO/NO⁻ donating compound is Piloty's acid. Alsodisclosed herein is a method of treating CHF by administering atherapeutically effective dose of at least one HNO/NO⁻ donating compoundin combination with a therapeutically effective dose of at least onepositive inotropic agent to a subject experiencing heart failure. Inparticular embodiments the HNO/NO⁻ donating compound is adiazeniumdolate, such as IPA/NO, and the positive inotrope is abeta-adrenergic agonist, such as dobutamine. Additionally, in particularembodiments of the methods described above, the HNO/NO⁻ donatingcompound or the combination of the HNO/NO⁻ donating compound and thepositive inotropic compound are used to treat Class III CHF, or othernon-acute CHF. In still other embodiments the methods are used to treatacute CHF. Also disclosed is a method of treating CHF in a subjectreceiving beta-antagonist therapy by administering a therapeuticallyeffective dose of at least one HNO/NO⁻ donating compound. In particularembodiments the HNO/NO⁻ donating compound is a diazeniumdolate, such asAngeli's salt.

A nitroxyl donor is an agent or compound that provides a physiologicallyeffective amount of HNO or NO⁻ (HNO/N⁻). The HNO/NO⁻ donating compoundis any compound that donates HNO/NO⁻ and has a safety profile indicatingthe compound would be tolerated by a subject in the amount necessary toachieve a therapeutic effect. One of ordinary skill in the art would beable to determine the safety of administering particular compounds anddosages to live subjects. Such a compound includes any compound havingthe formula

wherein J is an organic or inorganic moiety, M^(+x) is apharmaceutically acceptable cation, wherein x is the valence of thecation, a is 1 or 2, b and c are the smallest integers that result in aneutral compound, and wherein the compound is administered underconditions that cause it to release HNO/NO⁻. The compounds of Formula Iare known generally as diazeniumdolates because they contain theN-oxy-N-nitroso complex. Angeli's salt is a compound of formula I thatdisassociates under physiological conditions to donate HNO/NO⁻. Otherdiazeniumdolates that disassociate under physiological conditions togenerate HNO/NO⁻, such as IPA/NO or Sulfi/NO(N-nitrosohydroxylamine-N-sulfonate/ammonium salt), are also used inperforming the method. Additionally, analogs and derivatives of suchcompounds can be used. Moreover, conditions, such as the oxidation stateof the environment, can be altered to cause such compounds to donateHNO/NO⁻.

An analog is a molecule that differs in chemical structure from a parentcompound, for example a homolog (differing by an increment in thechemical structure, such as a difference in the length of an alkylchain), a molecular fragment, a structure that differs by one or morefunctional groups, or a change in ionization. Structural analogs areoften found using quantitative structure activity relationships (QSAR),with technologies such as those disclosed in Remington: The Science andPractice of Pharmacology, 19^(th) Edition (1995), chapter 28. Aderivative is a biologically active molecule derived from the basestructure.

Wang et al., “New chemical and biological aspects of S-nitrosothiols,”Curr. Med. Chem., 7(8):821-34 (2000), describes NO⁻ formation fromheterolytic decomposition of S-nitrosothiol compounds. Thus,S-nitrosothiol compounds such as S-nitroso-L-cystine ethyl ester,S-nitroso-L-cystine, S-nitroso-glutathione, S-nitroso-N-acetyl-cystine,S-nitroso-3-mercaptoetanol, S-nitroso-3-mercaptopropanoic acid,S-nitroso-2-aimonethanethiol, S-nitroso-N-acetyl penicillamine (SNAP),S-nitrosocaptopril, as well as others are also used in performing theprovided method. In particular, S-nitrosoglutathione (GNSO) has beenreported as capable of being reduced to HNO/NO⁻ in the presence ofthiols. Hogg et al., Biochem. J., 323:477-481 (1997).

Piloty's acid (benzenesulfohydroxamic acid) is a hydroxamic acid(X(═O)NHOH) that donates HNO/NO⁻ and is useful in performing theprovided methods. Other hydroxamic acids that donate HNO/NO⁻, inparticular, other sulfohyrdroxamic acids and their derivatives are alsouseful.

Thionitrates (R—(S)—NO₂, wherein R is a polypeptide, an amino acid, asugar, a modified or unmodified oligonucleotide, a straight or branched,saturated or unsaturated, aliphatic or aromatic, substituted orunsubstituted hydrocarbon, or a heterocylclic group) that donate HNO/NO⁻are useful in performing the methods provided. In particular, suchcompounds that form disulfide species are useful.

One of ordinary skill in the art would be able to determine these andother compounds capable of donating HNO/NO⁻. Also included in this termis direct administration of HNO/NO⁻.

Compositions comprising more than one HNO/NO⁻ donating compound are alsoused. For example, IPA/NO and another compound that dissociates togenerate HNO/NO⁻ for example, Piloty's acid, are used to treat heartfailure.

In particular embodiments the HNO/NO⁻ donating compound is administeredin the form of a pharmaceutical composition. A pharmaceuticalcomposition comprising an effective amount of the HNO/NO⁻ donatingcompound as an active ingredient could be easily prepared by standardprocedures well known in the art, with pharmaceutically acceptablenon-toxic solvents and/or sterile carriers, if necessary. Suchpreparations are administered orally or in injectable form, or directlyto myocardial tissue. In other embodiments the HNO/NO⁻ donor isadministered without a pharmaceutical carrier. In particular embodimentsthe HNO/NO⁻ donor is administered by a short-term infusion, such as for5 to 20 minutes. In other embodiments the HNO/NO⁻ donor is administeredby a long-term infusion, such as from 3-4 hours. The HNO/NO⁻ donated byAngeli's salt retains its beneficial effects during 3-4 hours ofperfusion.

The dose of the HNO/NO⁻ donating compound is a therapeutically effectivedose. A therapeutically effective dose of an HNO/NO⁻ donating compoundcomprises a dose effective to increase contractility in a subjectexperiencing heart failure. Optimizing therapy to be effective across abroad population can be performed with a careful understanding ofvarious factors to determine the appropriate therapeutic dose, in viewof the inventors' disclosure that these agents cause a positiveinotropic effect as well as venous dilation. In particular embodiments,an infusion of 10 micrograms (μg)/kilogram of body weight (kg)/minute(min) is administered for 5-20 min to treat acute heart failure. -In-oneexample,-the agent administered at this dose is Angeli's salt. In otherembodiments an infusion of 2.5 μg/kg/min is administered for 5-20 min totreat acute heart failure. In one example, the agent administered atthis dose is IPA/NO.

A positive inotrope is an agent or compound that causes an increase inmyocardial contractile function. Such an agent includes abeta-adrenergic receptor agonist, an inhibitor of phophodiesteraseactivity, and calcium-sensitizers. Beta-adrenergic receptor agonistsinclude, among others, dopamine, dobutamine, terbutaline, andisoproterenol. Analogs and derivatives of such compounds are also used.For example, U.S. Pat. No. 4,663,351 describes a dobutamine prodrug thatcan be administered orally. One of ordinary skill in the art would beable to determine these and other compounds that are capable of causingpositive inotropic effects and also additional beta-agonist compounds.In particular embodiments the beta-receptor agonist is selective for thebeta-1 receptor. However, in other embodiments the beta-agonist isselective for the beta-2 receptor, or is not selective for anyparticular receptor. Additionally, compositions comprising more than onepositive inotropic agent are used. For example, dobutamine andisoproterenol are used to treat heart failure.

In particular embodiments the positive inotropic agent is administeredin combination with the HNO/NO⁻ donor. The combined administration ofthe HNO/NO⁻ donor and the positive inotropic agent comprisesadministering the HNO/NO⁻ donor either sequentially with the positiveinotropic agent for example, the treatment with one agent first and thenthe second agent, or administering both agents at substantially the sametime, wherein there is an overlap in performing the administration. Withsequential administration a subject is exposed to the agents atdifferent times, so long as some amount of the first agent, which issufficient to be therapeutically effective in combination with thesecond agent, remains in the subject when the other agent isadministered. Treatment with both agents at the same time can be in thesame dose, such as a physically mixed dose, or in separate dosesadministered at the same time.

In particular embodiments the positive inotropic agent is administeredin the form of a pharmaceutical composition. A pharmaceuticalcomposition comprising an effective amount of the positive inotropicagent as an active ingredient could be easily prepared by standardprocedures well known in the art, with pharmaceutically acceptablenon-toxic solvents and/or sterile carriers, if necessary. Suchpreparations are administered orally or in injectable form, or directlyto myocardial tissue. In other embodiments the positive inotropic agentis administered without a pharmaceutical carrier.

The dose of the positive inotropic agent is a therapeutically effectivedose. In particular embodiments positive inotropic agent is administeredat a dose of between 2 and 20 μg/kg/min. In certain examples dobutamineis administered at this dose. However, in other embodiments, higher andlower dosages are administered to subjects experiencing heart failure.For example, a dose of 0.5 μg/kg/min is administered, or a dose of 40μg/kg/min is administered. Optimizing therapy to be effective across abroad population can be performed with a careful understanding ofvarious factors to determine the appropriate therapeutic dose, in viewof the inventors' disclosure that the positive inotropic agent isadministered in combination with an HNO/NO⁻ donor.

In particular embodiments an HNO/NO⁻ donor is administered to a subjectexperiencing heart failure that is receiving beta-antagonist therapy. Abeta-antagonist (also known as a beta-blocker) includes any compoundthat effectively acts as an antagonist at a subject's beta-adrenergicreceptors, and provides desired therapeutic or pharmaceutical results,such as diminished vascular tone and/or heart rate. In particularembodiments the beta-antagonist is selective for a particular receptor,such as the beta-1 receptor. In other embodiments the beta-antagonist isnot selective for any particular beta receptor. Beta-antagonizing agentsinclude metoprolol, bisoprolol, bucindolol, carvedilol, timolol,propranolol, pindolol, and atenolol. One of ordinary skill in the artwould be able to identify these and other compounds that are capable ofacting as beta-adrenergic antagonists at a subject's beta-adrenergicreceptors.

A subject who is receiving beta-antagonist therapy is any subject towhom a beta-antagonist has been administered, and in whom thebeta-antagonist continues to act as an antagonist at the subject'sbeta-adrenergic receptors. In particular embodiments a determination ofwhether a subject is receiving beta-blocking therapy is made byexamination of the subject's medical history. In other embodiments thesubject is screened for the presence of beta-blocking agents by chemicaltests, such as high-speed liquid chromatography as described in Theviset al., Biomed. Chromatogr., 15:393-402 (2001).

The administration of an HNO/NO⁻ donating compound either alone, incombination with a positive inotropic agent, or to a subject receivingbeta-antagonist therapy, is used to treat heart failure of allclassifications. In particular embodiments an HNO/NO⁻ donating compoundis used to treat early-stage chronic heart failure, such as Class IIheart failure. In other embodiments an HNO/NO⁻ donating compound is usedin combination with a positive inotropic agent, such as isoproterenol totreat Class IV heart failure. In still other embodiments an HNO/NO⁻donating compound is used in combination with a positive inotropicagent, such as isoproterenol to treat acute heart failure. In someembodiments, when HNONO⁻ is used to treat early stage heart failure, thedose administered is lower than that used to treat acute heart failure.In other embodiments the dose is the same as is used to treat acuteheart failure.

The following are non-limiting examples of particular embodiments of themethods provided herein.

EXAMPLE 1

This example demonstrates that infusion of an HNO/NO⁻ donor causedpositive inotropic effects in failing myocardium. Further, infusion ofan HNO/NO⁻ donor complemented the positive inotropic effect ofdobutamine, as opposed to the impairment of dobutamine's positiveinotropic effect observed with NO· donors. Additionally, when comparedwith an infusion of Angeli's salt designed to cause a systemic bloodpressure decrease nearly equivalent to that caused by IPA/NO, theHNO/NO⁻ donor IPA/NO exerted a stronger positive inotropic effect.

The effect of HNO/NO⁻ donated by AS (10 micrograms (μg)/kilogram(kg)/minute (min) for 5-20 min) and IPA/NO (2.5-5.0 μg/kg/min for 5-20min) on basal cardiovascular function was tested in mongrel dogs.Studies were performed at a constant heart rate during atrial pacing(130-160 beats per minute). Myocardial effects produced by HNO/NO⁻donating compounds were compared to those produced by the NO· donorsDEA/NO and nitroglycerin at doses titrated to achieve the same declinein systolic pressure (a measure of systemic blood pressure) as theHNO/NO⁻ donors.

Hemodynamic data was sampled at 250 Hertz (Hz) and steady-state andpressure-dimension parameters were derived. Since in vivo cardiaccontractility assessment requires separation of the effects of chamberloading, pressure-volume relation indexes, specifically, theend-systolic elastance (Ees), and the slope of dP/dt_(max)-end-diastolicdimension (D_(EDV)) relations were employed. Isovolumic relaxation wasderived from pressure decay waveforms assuming a nonzero decayasymptote.

Serum concentrations of nitrite and nitrate were determined by amodified Griess assay, with and without prior chemical reduction ofnitrate to nitrite using VCl₃. Serum stored at −70° C. was deproteinizedby ultrafiltration (30 kilodalton (kD) cut-off, Centricon, Sartorius) at4° C., and absorbance at 540 nanometer (nm) read using a plate reader(Perkin Elmer HTS 7000 BioAssay Reader controlled by TECAN WinSelectsoftware) after a 37° C. incubation with Griess reagents for 30-45 min.

With reference to FIG. 1, each compound tested was administered in dosestitrated to achieve nearly equivalent end systolic pressures (Pes) inorder to allow comparison between equivalent levels of dilation.Angeli's salt and IPA/NO caused significant increases in contractilityduring heart failure as measured by Ees, D_(EDV), and PRSW. Theseincreases were much greater than the small increases observed withDEA/NO and were opposite of the negative inotropic effects observed withnitroglycerin. Additionally, both Angeli's salt and IPA/NO reduced thecardiac load as measured by Edv (preload) and Ea (afterload).Surprisingly, IPA/NO caused a greater increase in cardiac contractilitythan Angeli's salt as measured by Ees, which, being load-independent, isa good parameter for assessing myocardial contractility. This isespecially surprising because the doses of IPA/NO were one-half toone-quarter the doses of Angeli's salt.

With reference to FIG. 2, the administration of HNO/NO⁻ exhibited apositive inotropic effect, which was not dependent on cardiac load. Asillustrated by the measurements of Ees and PRSW for both loaded andunloaded states, HNO/NO⁻ exerted a nearly equivalent positive inotropiceffect regardless of cardiac load. This indicates that the contractilityincreases caused by HNO/NO⁻ are primary as opposed to secondary effects.In contrast, with reference to FIG. 3, the minor positive inotropiceffects observed with the administration of NO· (DEA/NO) were reversedwhen the heart was under cardiac load conditions, that is at matchedend-diastolic volume. Moreover, FIG. 4 illustrates that administrationof the NO· donor nitroglycerin caused contractility to decrease whenadministered alone, and caused an even greater negative inotropic effectunder loading conditions. This indicates that the minor contractilityincrease observed with DEA/NO is merely secondary to the vasodilatoryeffects of the compound. That is, NO· has no direct positive inotropiceffects because any increases in contractility were abolished uponvolume repletion.

With reference to FIG. 5, administration of AS and IPA/NO resulted in agreater positive inotropic effect than administration of dobutaminealone. For example, administration of AS resulted in a more thandoubling of Ees over administration of dobutamine alone. In contrast,administration of DEA/NO and nitroglycerin reduced the positiveinotropic effect of dobutamine, as illustrated by the decrease in Eeswhen the dobutamine was administered with DEA/NO and NTG.

EXAMPLE 2

This example demonstrates that the positive inotropic effect of HNO/NO⁻is a function of its stimulation of calcitonin gene-related peptide(CGRP) signaling rather than a function of beta-agonism.

To test the relation between the inotropic action of HNO/NO⁻ andcalcitonin gene-related peptide (CGRP) signaling, CGRP receptors inmongrel dogs were antagonized using the selective antagonist CGRP₈₋₃₇(400 μg in 30 milliliters (ml) of saline bolus, then 2.6 μg/kg/min for15 min). Plasma CGRP levels measurements were performed by sampling theblood of the dogs. Blood samples (2.5 ml) were withdrawn from arterial,venous, and coronary sinus catheters. After sampling, catheters wereflushed with heparanized saline. Samples were centrifuged at 1600 timesgravity (g) for 20 minutes at 4° Celcius (C). Plasma was then separatedand stored at −20° C. until analysis. Plasma (0.5 ml) was used toextract CGRP by addition of 0.8 ml of ethanol. The mixture wascentrifuged at 1600 g for 20 minutes. After removing the supernatant,the extracted samples were air dried at room temperature overnight andthen stored at 4° C. Immediately prior to assay, dried samples werereconstituted with assay buffer following manufacturer's instructions(Peninsula Labs) and assayed for CGRP by radioimmunoassay (RIA). CGRPantiserum, code RAS 6012, was used. The dynamic assay range was 1-128picograms (pg) per 300 microliters (μL ) of sample. Stimulation withHNO/NO⁻ donors and diagnostic tests were performed as described above inExample 1.

With reference to FIG. 6, administration of the selective CGRPantagonist CGRP₈₋₃₇ resulted in a modest negative inotropic effectduring heart failure as measured by Ees. This result was not unexpectedgiven that CGRP is known positive inotrope. Doggrell, Expert Opin.Investig. Drugs, 10:1131-8 (2001). More interestingly, CGRP₈₋₃₇effectively prevented the HNO/NO⁻-mediated, positive inotropic effect ofAngeles salt as is illustrated by a comparison of the Ees data resultingfrom the combined administration of CGRP₈₋₃₇ and Angeli's salt with theresults observed from administration of Angeli's salt alone. Theseresults illustrate that the positive inotropy of HNO/NO⁻ is caused bystimulating release of CGRP, which is a nonadrenergic/noncholinergic(NANC) neuromodulator.

This is supported by the data illustrated in FIG. 7, which show thatblood plasma CGRP levels were increased by administration of the HNO/NO⁻donor Angeli's salt in both normal and heart failure conditions. Asensitive and specific radioimmunoassay (RIA) was used to study bloodplasma levels of CGRP in normal and in CHF dogs, both in basal andstimulated conditions (after administration of AS, DEA/NO andnitroglycerin). The basal mean plasma levels of CGRP were 23, 24.5 and27 pg/ml in the artery, vein, and coronary sinus of normal dogs,respectively. These levels were significantly reduced in all vascularcompartments in CHF dogs: 13.3±0.7, 14.3±1.4, and 14±0.6 pg/ml inartery, vein, and sinus, respectively. When stimulated with the HNO/NO⁻donor AS, plasma CGRP levels increased substantially in both normal andCHF dogs (FIG. 7). In contrast, stimulation with DEA/NO andnitroglycerin failed to significantly increased CGRP levels. These dataclearly show that HNO/NO⁻ directly stimulates the release of CGRP.

EXAMPLE 3

This example demonstrates that HNO/NO⁻ effectively increasescontractility even when administered to a subject receivingbeta-antagonist therapy.

As illustrated in FIG. 8, administration of the HNO/NO⁻ donor Angeli'ssalt (as described in Example 1) to a normal subject that is receivingbeta-antagonist therapy (propranolol, 2 milligrams/kg in bolus) causedan increase in contractility as indexed by Ees and D_(EDV). Thisincrease was observed despite the propranolol-induced reduction inmyocardial performance. Similar results were obtained in one heartfailure subject (data not shown).

The above-described examples merely provide particular embodiments ofthe provided method. They are not intended to be limiting in any way.Moreover, although embodiments of the method provided have beendescribed herein in detail, it will be understood by those of skill inthe art that variations may be made thereto without departing from thespirit of the invention or scope of the appended claims.

1. A method of treating heart failure comprising, Administering to asubject experiencing heart failure, a therapeutically effective dose ofat least one nitroxyl donating compound and at least one positiveinotropic compound, wherein the does is effective to increase myocardialcontractility. 2-25. (canceled)